The overall goal of this AREA renewal application is to develop microchip approaches that can be used to study cell-to-cell interactions at the molecular level. There are many examples in which the interaction of different cell types plays a role in normal biological function or in the onset of disease. One example is the interaction between neurons and glia cells that have undergone inflammation. It has been suggested that the degeneration of dopaminergic neurons that is prevalent in Parkinson's disease may be related to chronic inflammation of microglia, with the microglia producing nitric oxide and other reactive oxygen species that interact with the neurons. Another example is the vasodilatation process, where it has been shown that red blood cells, when exposed to hypoxic conditions or deformation, interact with endothelial cells via ATP release, leading to the production of nitric oxide and subsequent smooth muscle relaxation. Impairment of the ATP release from red blood cells leads to less nitric oxide production and has been postulated to play a role in several diseases. While there have been numerous examples of immobilizing cells on-chip and detecting a specific analyte released from the cells, there have been few reports of integrating multiple cell types on chip where cell-to-cell communication can be studied in a manner where numerous neurotransmitters/products can be monitored. In this grant we propose to continue the development of microchip approaches that enable the integration of cell immobilization with a general analysis step (electrophoresis), where a variety of analyses that are either released or transported through a layer of cells can be separated and subsequently detected via electrochemistry. Importantly, we propose to develop technology that will enable a researcher to monitor cell- to-cell communication between 2 layers of immobilized cells (PC 12 cells and microglia cells) or between a flowing stream of cells (red blood cells) and a layer of immobilized cells (endothelial cells). In addition, we propose to expand this microchip approach to enable in vivo studies by integrating microdialysis sampling, which can be used to study the interactions of different cell types by stereotaxically implanting a probe in the region of interest, with segmented flow, microchip electrophoresis, and electrochemical detection. The specific aims of the grant are to 1) Use of reservoir-based cell immobilization and microchip electrophoresis with electrochemical detection to study the interaction between multiple cell types;2) Develop a microchip device that can be used to study cell-to-cell communication between red blood cells and an immobilized endothelium;and 3) Integration of microdialysis sampling and segmented flow with microchip electrophoresis and electrochemical detection for in vivo sampling. PUBLIC HEALTH RELEVANCE: The overall goal of this AREA renewal application is to develop microchip approaches that can be used to study cell-to-cell interactions at the molecular level. The resulting technology will enable monitoring of cell-to- cell communication between 2 layers of immobilized cells or between flowing cells and an immobilized endothelium. The approaches will also enable in vivo studies by integrating microdialysis sampling with segmented flow, microchip electrophoresis, and electrochemical detection.